High performance liquid chromatography method and apparatus

ABSTRACT

High pressure liquid chromatographic apparatus in which a fluid mixture containing at least one solute that is reactive with chromatographically reactive surfaces in the column is loaded into the column, and a number of plugs of different eluant fluids are injected into that column. The injections are made in a manner that minimizes the amount of eluant required. In one embodiment, the injections are made to insure that flow of at least the eluant fluids through the column will occur, preferably with a substantially flat wave front, at speeds corresponding to reduced velocities greater than about 5,000, i.e. at flow rates sufficient to induce turbulent flow in those fluids, thereby minimizing the time required for the entire succession of mixture and eluant fluids to traverse the column. In other embodiments, the injections are made substantially simultaneously at spatially separated points adjacent the entrance to the column or are made in sequence to a single location adjacent the entrance to the column, in either case the fluids then tend to travel through the column at a common group velocity as closely bunched fluid plugs.

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/588,874, filed Jan. 19, 1996, now abandoned, and of U.S.provisional patent application Ser. No. 60/027,216, filed Sep. 30, 1996.

This invention relates generally to chromatography and more particularlyto methods and procedures for effecting improved high performance liquidchromatography (HPLC).

BACKGROUND OF THE INVENTION

The separation process effected through high performance liquidchromatography relies on the fact that a number of component solutemolecules in a sample stream of a fluid (known as the mobile phase)flowing through a packed bed of particles (known as the stationaryphase) usually in a column, can be efficiently separated from oneanother. Basically, because each individual sample component has adifferent affinity for the stationary phase, each such component has adifferent rate of migration through and a different exit time from thecolumn, effecting separation of the components. The separationefficiency is determined by the amount of spreading of the solute bandas it traverses the bed or column.

Such separations, particularly in preparative processes, havesignificant limitations typically occasioned by the batch nature of theprocesses. Typically, a chromatographic column is first equilibrated byflowing an equilibrating fluid through the column, the latter is thencharged or loaded with a fluid mixture containing the solute or solutessought to be separated, and one or more eluant fluids are flowedsequentially through the column to release bound solute selectively. Theeluted solutes are thus temporally separated in the flowstream emergingat the output of the column and the process may be repeated cyclically.At typical flow rates and where the concentration of the desired solutesin the initial fluid mixture are low, the production of significantquantities of the desired solutes can be an unhappily slow and veryexpensive process with current apparatus and methods that requirecomparatively long cycle times.

A chromatography system of the prior art, as shown schematically in FIG.1, typically includes chromatographic column 20 the input of which isusually fed from a plurality of reservoirs provided for storing at leasta corresponding plurality of different fluids. Thus, reservoir 22 servesto store a supply of equilibrating fluid 24, while reservoirs 28 and 26respectively store corresponding supplies of eluant fluid 32 and fluidmixture 30 containing the sample to be separated. Pump 34 is usuallycoupled between the proximal or input end of column 20 and a pluralityof valves means 36, 37 and 38 respectively connected to the outputs ofreservoirs 22, 26 and 28, pump 34 serving to force fluid from thevarious reservoirs into column 20. Disposed adjacent the distal oroutput end of column 20 is the usual detector 39 of any type well knownin the prior art.

In operation, column 20 is usually first equilibrated by opening valvemeans 36 and running pump 34 so as to permit fluid from reservoir 22 tobe pumped at a predetermined flow rate through column 20 to equilibratethe latter. Then, typically valve means 36 is closed and valve means 37is opened to permit a quantity of fluid mixture 30 to be pumped intocolumn 20, loading the latter with solute molecules that bind tochromatographically reactive surfaces located within column 20. Lastly,valve means 36 and 37 are closed and valve means 38 is opened to permiteluant fluid 32 from reservoir 28 to be pumped through the loadedcolumn. The solute molecules eluted from the column by the eluant fluidare detected, typically optically by detector 39, and may be separatedin a known type of fraction collector 35.

The use of several reservoirs for the equilibrating fluids and eluants,all being fed alternatively into the column by a pump through extensiveconduit and valve systems, leads to mixing of the various fluids,contributes to band spreading, is wasteful of often expensive eluantfluid, and introduces undesirable delays in operation occasioned byvalve switching and the necessity of transferring the volume ofunexpended fluids temporarily stored within the conduits and valves.Such delays until very recently have been considered negligible comparedto the relatively long cycle time in the column. There is also theinevitable mixing that occurs when the various fluids are introducedinto and expelled from the same pump, both the delays and the mixingtending to promote band-broadening and reduce efficiency. The timerequired ordinarily between the introduction of sample into the columnand the ultimate separation of the sample components at the columnoutput can readily exceed many hours and often days.

A major problem impeding speed and throughput of separations in priorart HPLC systems arises out of the use of chromatography columnsoperating under the constraints imposed by the well-known Van Deemterequations and the consequent arrangement of the physical components ofthe system.

Because chromatographic system obeying the Van Deemter equations arebelieved to operate with substantially laminar fluid flow, the fluidwave fronts of the different fluid flows into the column tend to assumeparaboloidal configurations, thereby precluding sharp separationsbetween volumes of different fluids traversing the column, contributingto greater mixing. It has recently been discovered that the limitationsimposed on such HPLC separations by operation at a mobile phase flowrate dictated as optimal by the Van Deemter curves can be overcome bynovel methods of performing liquid chromatography employing an eluantflow rate through the chromatography column at a speed corresponding toan average reduced velocity (as hereinafter defined) greater than about5000. It is believed that under such conditions, turbulent flow of theeluant is induced within the column and it is postulated that suchturbulent flow enhances the rate of mass transfer, thus increasing thethroughput/productivity of the column by reducing dramatically the timerequired to effect separations. These novel methods of and apparatus forperforming liquid chromatography are described more fully in U.S. patentapplication Ser. No. 08/552193 filed Nov. 2, 1995, now abandoned, thesame being incorporated in its entirety herein by reference.

It has been customary to describe the function of an HPLC column in aplot in which column plate height H is plotted against linear velocity uof the mobile phase. Since an HPLC process is a diffusion-driven processand since different solute molecules have different diffusioncoefficients, one can consider this latter variable in applying theprocess to a wide range of solutes of different molecular weights.Additionally, the size of the particles in the column may differ fromcolumn to column, and may also be considered as another variable.Similarly, the viscosity of the solvent for the solute might beconsidered. In order to normalize the plots to take these variables intoaccount, one advantageously may employ reduced coordinates,specifically, h in place of H, and v in place of u, as taught byGiddings and described in Snyder & Kirkland Introduction to ModernLiquid Chromatography, 2nd Ed., John Wiley & Sons, Inc., (1979) atpp.234-235, to yield a reduced form of the Van Deemter equation asfollows:

    h=a+b/v+cv, or                                             (Equ. 1)

    H=ad.sub.p +bD/u+cud.sub.p /D                              (Equ. 2)

wherein a, b and c are coefficients, and the coordinate h is defined asH/d_(p), d_(p) being the particle diameter; accordingly h is adimensionless coordinate. Similarly, the dimensionless coordinate, thereduced velocity v, is defined as ud_(p) /D where D is the diffusioncoefficient of the solute in the mobile phase.

It will be recognized that v is also known as the Peclet number. Itshould be stressed, however, that the reduced coordinate or Pecletnumber, v, as used in the instant exposition of the present invention,is descriptive of fluid flow through the entire column, and should notbe considered as descriptive of fluid flow within the pores of porousparticles that may constitute a packed bed in the column.

OBJECTS OF THE INVENTION

A principal object of the present invention is to provide improvedchromatographic apparatus and processes for high productivity, highresolution separation of solutes, such as biologicals and the like.Other objects of the present invention are to provide such apparatus andprocesses as will dramatically enhance the throughput/productivity ofpreparative chromatography, to provide such apparatus and processeswhich are sparing of eluant fluids, and to provide such apparatus andprocesses in which separations are effected in relatively short timewithin the chromatographic column and in which minimized spatialseparation is maintained between bodies of fluids prior to introductioninto the column and while those traverse the column.

SUMMARY OF THE INVENTION

To these ends the present invention is directed to novel methods of andapparatus for performing liquid chromatography wherein at least onesolute mixture sample is loaded onto a chromatography column and issubsequently eluted from the column by one or more eluant fluids. Atleast the sample mixture and one or more eluant fluid or fluids areintroduced into the column, preferably by rapid injection, at one ormore locations adjacent the proximal or input end of the column so thatthe fluid injections form closely bunched but separated fluid plugs. Thefluids may be injected at any reasonable flow rate, but are preferablyinjected into the column so as to traverse the latter at a high flowrate, preferably at a speed corresponding to an average reduced velocitygreater than approximately 5000. The fluids are thus fed directly intothe column without any intervening common pump or other device thatmight tend to promote undesirable mixing prior to introduction of thefluids into the column, and thereafter when in the column in such manneras to maintain minimized spatial step separation between the plugs asthe latter traverse said column, even at a speed corresponding to areduced velocity greater than about 5000.

In one variation of the present invention, desired discrete plugs, ashereinafter defined, of the fluid mixture to be separated and one ormore eluant fluids are injected sequentially by separate injectors, theoutputs of which are all connected to a common input port located at theinput of the column and each of which injectors can be operated toswitch injection of fluid plugs rapidly in and out of the main stream offluid running from the main pump.

Yet another variation of the present invention involves the use ofmultiple injectors each connected at a separate location spatiallyseparated from one another along the axis of elongation of thechromatographic column and operable to substantially simultaneouslyinject discrete plugs of the various fluids at the spatially separatedlocations.

The term "plug" as used herein in connection with injected volumes is tobe understood to mean a mass or volume of fluid that is injected into aflowstream in a chromatographic column so as to form a discrete,essentially isomorphic mass extending substantially completely acrossthe column and preferably having approximately flat or planar front andrear surfaces extending perpendicular to the axis of elongation of thecolumn.

The foregoing and other objects of the present invention will in part beobvious and will in part appear hereinafter. The invention accordinglycomprises the apparatus possessing the construction and arrangement ofparts exemplified in the following detailed disclosure, and the methodcomprising the several steps and the relation and order of one or moreof such steps with respect to the others, the scope of the applicationof which will be indicated in the claims.

For a fuller understanding of the nature and objects of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the drawings wherein line numerals denote likeparts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified schematic diagram of a typical prior artapparatus;

FIG. 2 is a schematic diagram of apparatus embodying the principles ofthe present invention;

FIG. 3 is an enlarged schematic view in cross-section of achromatographic column employed in the apparatus of FIG. 2;

FIG. 4 is a schematic representation of a typical dual-coil injectoruseful in the present invention and shown in a first state;

FIG. 5 is a schematic representation of the dual-coil injector of FIG.4, in a second state;

FIG. 6 is a schematic representation of a single coil injector useful inthe present invention and shown in a first state;

FIG. 7 is a schematic representation of the injector of FIG. 6, in asecond state; and

FIG. 8 is a schematic representation similar to the embodiment of FIG.2, but wherein all injectors feed a single input to the chromatographiccolumn.

FIG. 9 is a schematic representation of an embodiment of an apparatusembodying the principles of the present invention.

FIG. 10 is a schematic representation of an embodiment of an apparatusembodying the principles of the present invention.

FIG. 11 is a schematic representation of an embodiment of an apparatusembodying the principles of the present invention.

FIG. 12 is a schematic representation of an embodiment of an apparatusembodying the principles of the present invention.

FIG. 13 is a chromatogram illustrating a preparative separation usingthe apparatus of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention, as shown in FIGS. 2 and 3, isembodied in chromatography apparatus comprising a chromatographic column40 formed as a packed multiplicity of rigid, solid particles 42 havingsubstantially uniform mean diameters of not less than about 30 μm. Theterm "mean diameter" as used herein is intended to mean the average(mean) diameter or cross-section dimension of the particles regardlessof particle configuration and is not to be construed as limited toparticles that are necessarily spherical or regular solids, the value ofsuch mean diameter typically being within a distribution of diameters ata confidence coefficient of about 95%. A preferred aspect of the presentinvention is the irregularity of particles' shape. The term "irregular"as used herein is intended not only to be defined as lacking conformityof form, inasmuch as the particles can be present in a mixedmultiplicity of various polyhedral configurations, but is intended toinclude solids of revolution such as generally spherical, conoidal,ellipsoidal and the like type of particles with rough, uneven orscabrous surfaces.

The particles used in the aforesaid embodiment of the present inventionare formed from materials that are incompressible, which term is to beunderstood to mean that the time rate of changes of the densities andvolumes of the particles under pressures of at least about 5×10³ psi,(including outlet column frit retainer) remains substantially zero, andthe particles therefore will substantially resist plastic deformationeven at such high pressure. The particles of the present invention areshaped and selected in a range of sizes and shapes such that they can bepacked at a pressure sufficient to form a column characterized in havinginterstitial channels 44, as shown particularly in FIG. 3, formedbetween particles 42. Because of the irregularity of the particles, itwill be recognized that the interior walls of such channels arenecessarily quite rough in configuration. While it is believed that atleast the majority of channels 44 have mean cross-section diameterssubstantially not less than about 4 μm, the interstitial volume fraction(i.e. the total volume of interstitial channels 44 between theparticles) should not be less than about 45% of the total volume ofcolumn 40. It will be appreciated that typical columns of the prior arthave interstitial volume fractions less than about 45%, moreparticularly ranging from about 35% to 42%. The surfaces of particles 42are chromatographically active either per se as is well known in theart, or by treatment, as by coating, with any of the many knownchromatographically active, stationary phase layers, also as well knownin the art.

Particles 42 may be pellicular, or to increase active surface area, maybe porous with the intraparticle pores typically having mean diameterslying within a range of about 60 Åto 5,000 Å. As a result of theparticle irregularity, coupled with an interstitial volume fraction ofnot less than about 45%, it is believed that turbulent flow through theinterstitial channels of the column of the present invention cansurprisingly be induced at Reynolds numbers well below 10.

In the present invention, means, such as pump 46 coupled to the proximalend of column 40, is provided for pumping a fluid, such as a firstequilibrating fluid 47 from an appropriate source such as reservoir 48,flowing through at least a major portion of the interstitial volume incolumn 40, preferably at a reduced velocity (i.e., ud_(p) /D asabove-defined) substantially above about 5000. The latter is anapproximate value at which the slope of the h/v curve begins to decreasealong the reduced coordinate h (i.e. H/d_(p)) axis, indicating animprovement in efficiency with increasing reduced velocity. It isbelieved that turbulent flow of the mixture is induced in the column ofthe present invention at a flow velocity corresponding to a reducedvelocity value of about 5000.

The present invention further includes means, such as loop injector 50for injecting plugs of fluid sample mixture 52 into column 40.Typically, where the present invention is being employed for preparativepurposes, the plug of solute mixture 52 will be as large as practicableto fully load column 40. Mixture 52 contains the solute or solutes ofinterest, and is pumped into injector 50 by auxiliary pump 54 fromanother appropriate reservoir or storage tank 56.

The present invention also includes means, such as at least one loopinjector 58 for flowing eluant fluid 60 into column 40. Fluid 60 ispumped into injector 58 by another auxiliary pump 62 from anotherappropriate reservoir or storage tank 64. In the embodiment shown inFIG. 2, yet another loop injector 66 is also shown for injecting asecond eluant fluid 68 into column 40. The latter is fed into injector66 by a third auxiliary pump 70 from another appropriate reservoir orstorage tank 72. It will be appreciated that pumps 46, 54, 62 and 70 maybe any desired type of known pump and are not to be limited tomechanical pumps, but may be any known system for imposing pressure onthe respective fluid to cause the latter to flow at the desired flowrate. It will be understood that additional injectors for other eluantfluids may also be provided as desired. For example, FIGS. 10 through 12depict embodiments that include additional eluant fluids 120, 124, and128, in reservoirs 122, 126, and 130, respectively, in a variety ofconfigurations to allow optimal flexibility in delivery of a particulareluant fluid to the flow path of the chromatographic system of theinvention.

Any injection means may be employed to apply sample and eluant fluids tocolumn 40 in accordance with the present invention, so long as the meansallows a measured amount of fluid to be delivered into the flow path.For example, the injection means may include a loop injector or asection of tubing, in combination with a pump means. Alternatively, theinjection means may be driven by the action of gravity applied to theflow path.

In the embodiment shown in FIG. 2, input conduit 74 is coupled from theoutput of pump 46 to input end 75 of column 40, and injectors 50, 58, 66are all connected to input conduit 74 at different locations spacedapart from one another between pump 46 and input end 75 of column 40.Operation of each injector can be manually controlled or automaticallycontrolled to inject respective plugs of sample mixture 52 and eluantfluids 60 and 68 into the proximal end of column 40, preferably atspeeds corresponding to reduced velocities above about 5000. Inaccordance with the invention, manually controlled operation of theinjectors encompasses operation mediated by human intervention, eitherto perform loading of the injector, to perform injection of the sampleor eluant fluid into the flow stream of the system into the column, orto perform both aspects of the injection process. Automaticallycontrolled operation of injectors is defined herein as operation whichdoes not require human intervention to perform loading of injectorsand/or injection of sample or eluant fluid into the flow stream of thesystem into the column. In the preferred embodiment, the automaticallycontrolled injectors are operated by actuators such as motors which areoperated under the control of a computer system. An automaticallycontrolled injector of the invention may also include a manuallyoperated injector as a backup to the automatically controlled injector.The apparatus of the invention may optionally include both manuallycontrolled and automatically controlled injectors.

The injectors may be operated to inject the respective fluid plugssubstantially simultaneously or in a timed sequence. For example, wherethe injection coil, as hereinafter described, in injector 50 is sized todeliver a 100 mL plug, the corresponding coils in injectors 58 and 66may be sized to provide plugs of, for example, 5 mL each of eluantfluids, and generally would have injection coils with correspondinglysmaller volumes. In such case, it is desirable to place sample injector50 at a location more remote from the input end of column 40 than theother injectors to avoid having to inject the small amounts of eluantinto column 40 through the large diameter coil conduit in injector 50and avoid the consequent mixing that would tend to occur. FIG. 10depicts a configuration of the chromatography apparatus of the inventionin which the sample injection means 100 and eluant injection means 102,104, 106, 108, and 110 are connected in series, and the sequence ofoperation of the various injection means is controlled by flow switchingvalve means 82, 84, 86, 88, 90, and 92. In FIG. 11, sample injectionmeans 100 and eluant injection means 102, 104, 106, 108, and 110 areconnected in such a way that sequential delivery of the various fluidsto the flow path is accomplished, under the control of flow switchingvalve means 82, 84, 86, 88, 90, and 92. The embodiment of FIG. 12depicts a configuration that allows delivery of a second equilibratingfluid 47' to the flow path from reservoir 48', through the action ofpump means 46'. Valve means 80 operates upstream of flow switching valvemeans 82, 84, 86, and 88, for example, to apply an elution gradient tothe flow path. It will be apparent that the spacing and location of thesample and eluant injectors relative to the column and any input conduitthereto may be other than that shown in the drawings.

Injection in preparative LC is particularly important inasmuch asloading of the sample across the entire column cross-section isgenerally preferred as permitting better use of the total columnpacking. In the present invention, sample inlet distributing head 76 ispositioned at the input end of column 40 and employed to distribute theinjected volumes as plugs with preferably a substantially flat or planarsurface or front perpendicular to the axis of elongation of the column,not only for the foregoing reasons but and also to maintain a relativelysharp surface of demarcation between adjacent plugs of fluid. Fluid isintroduced into head 76 at a pressure that will ensure that the plug orvolume injected into column 40 is moving at substantially the samevelocity as the flowstream generally, thereby minimizing mixing ofadjacent plugs of fluids and preserving the approximate planarity of theends of each plug where the plugs are adjacent to one another.

The plug of mixture 52 flowing through column 40 serves to load thelatter as solute molecules become bound to chromatographically activesurfaces in the column. The solute molecules eluted from the column bythe eluant fluid are detected, typically optically by detector 78, of atype and in a manner well known in the prior art, disposed at the distalend of column 40. For superior results, the eluant flow through column40 is preferably at a velocity corresponding to a reduced velocity ofabove about 5000, so that band spreading of solute eluted by the eluantfluid from the column in the present invention is an inverse function ofthe Reynolds number for the eluant fluid and a direct function of themagnitude of the diffusion coefficient of the solute in the eluantfluid. It should be understood, however, that the present invention isapplicable to chromatography in which the flowstreams through the columnare laminar inasmuch as the present invention tends to minimize theamount of eluant employed in either case.

In one embodiment of the present invention, column 40 is formed bypacking particles having a mean diameter not less than about 30 μm,preferably under pressure of at least about 5×10³ psi to insure that thecolumn formed will include substantially no voids except forinterstitial channels 44 formed between particles 42 in contact with oneanother, i.e. column 40 has a substantially uniform bulk density.Columns 40 formed in this manner, regardless of whether or not theparticles are porous or non-porous, should exhibit interstitialfractions of about 45% or higher. Lower interstitial fractions,typically around 35% for porous, non-rigid polystyrene particles, willnot exhibit the requisite reduced fluid velocity except at unacceptablyhigh pressure that will tend to collapse or rupture the particles.

In order to insure the formation of the desired uniform density columnwith the preferred interstitial fraction and preclude collapse underoperating pressure, the particles used to pack a column in the presentinvention are rigid solids that must necessarily be incompressible atpacking pressure of at least about 5×10³ psi, preferably up to pressuresas high as about 1×10⁴ psi. To that end, the preferred particles areformed from materials such as alumina, titania, silica, zirconia,vanadia, carbon and combinations thereof.

The method of the present invention therefore requires that the flowthrough at least a majority of the interstitial channels in thechromatographic column must be turbulent. It is postulated that theturbulent flow profile is almost flat, as distinguished from the typicalparabolic flow profile characteristic of laminar flow through achromatographic column. More importantly, it is believed that whenturbulent flow is induced, a radial component of velocity issuperimposed upon the normal diffusion process, altering the normaldiffusional process and the band-spreading kinetics in a favorablemanner with respect to the efficiency of the column. It is furtherpostulated that in order to induce and sustain turbulent flow throughthe column, there is a critical relationship between the diameter of theflowing channel and the linear flow velocity. The need for particlesthat are rigid and can withstand changes in pressure without plasticdeformation, as above-indicated, is therefore very important in suchcase.

Injectors useful in the present invention are commercially sold as, forexample, Model 3725 and Model 3725-038 manual injectors available fromRheodyne Incorporated of Cotati, Calif., Model E-45 automatic injectorsavailable from Valco Instrument Company, Inc. of Houston, Tex., andsimilar injectors provided by these and several other manufacturers. Aschematic diagram of a typical double-loop injector of such type,employing the symbols used by the manufacturer, is shown in twoalternative states in FIGS. 4 and 5. In each such Figure, the injectorincludes first input port 80 connected to the output of main pump 46,first injector loop coil 82, first output port 84 connected to input end75 of column 40, second input port 86 connected to the output ofauxiliary pump 54, second injector loop coil 88, and second output port90 which discharges to waste, and means for switching or valving thevarious input and output ports through different loop coils.

The operation of the injector of FIGS. 4 and 5 can be advantageouslydescribed in connection with injection of sample fluid from reservoir 56but it is to be understood that the description applies equally as wellto injection of eluant fluid from corresponding reservoirs. Thus, inoperation, it can be initially assumed that in the first state of theinjector as shown in FIG. 4, coil 82 is already filled with sample fluid52. In FIG. 4, the internal valving of the injector is shown asconnecting input port 80 to one side of coil 82, the other side of thelatter being connected to output port 84. Thus, as fluid pumped by pump46 is introduced abruptly, by the switching or valving action of theinjector, into and through port 80, the fluid pressure imposed serves tohydraulically force the sample fluid out of coil 82, replacing thesample fluid with fluid 47 from reservoir 48, and injecting the samplefluid from coil 82 through port 84 into column 40 as a plug. Thisswitching action of the injector also serves to connect input port 86 toone side of second loop coil 88, filling the latter as loop coil 82 isemptied of sample fluid, the other side of coil 82 being connected toport 90 so that any excess sample fluid is discharged to waste throughport 90.

As shown in FIG. 5, when the injector is switched to its second state,the internal valving of the injector is shown as connecting input port80 to one side of coil 88, the other side of the latter now becomingconnected to output port 84. Thus, as fluid pumped by pump 46 is againintroduced abruptly, by the switching or valving action of the injector,through port 80, the fluid pressure imposed serves to forcehydraulically the sample fluid contained in coil 88 out through port 84into column 40 as another plug. This switching action of the injectoralso serves to connect input port 86 to one side of first loop coil 82filling the latter with sample fluid again. The other side of coil 82becomes connected to output port 90 so that any excess fluid isdischarged to waste. The injection of plugs of fluid 52 into column 40are typically followed with injections of one or more plugs of eluantfluids 60 and 68, or by a flow of fluid 47 that will occur simply bypermitting a flow of the latter to proceed seriatim through one or moreof the several injectors.

While the structure and operation of the injectors has been described interms of double loop coil injectors, it will be understood that it isnot so limited. For example, as shown in FIGS. 6 and 7 wherein likenumbers denote like parts, single loop injectors with by-pass switchingcan also be employed. In FIGS. 6 and 7, all of the parts of theinjectors are identical to those of FIGS. 4 and 5 except that a by-passconduit 92 of minimal storage capacity is used to replace loop coil 82.It will be apparent that yet other known types of injectors can also beadvantageously employed in the apparatus of the present invention.

Switching operation of a plurality of injectors is preferably under thecontrol of means, such as a known computer or controller to optimizeswitching time and obtain the desired sequence of plug injections intocolumn 40. Injectors of the type described are preferred inasmuch asthey tend to provide desirably sharp transition boundaries betweenadjacent plugs of fluid, thus reducing mixing.

As shown in FIG. 2, injectors 50, 58 and 66 are coupled to input conduit74 to column 40 at spaced-apart locations, and it will be understoodthat in such case, all of those injectors can be operated simultaneouslyby means such as an appropriately programmed computer, to provide asuccession of spatially separated plugs presented to head 76 or may beoperated serially to provide even more flexibility in spacing thelocation of the plugs as the latter transit column 40. Yet, as shown inFIG. 8, wherein like numerals denote like parts, all of the injectorsmay be coupled directly to port 75 or a single location on conduit 74,and in such case, all of those injectors can be operated serially toprovide a succession of spatially separated plugs presented to head 76.

In the embodiment depicted in FIG. 9, solvent reservoir A is in fluidcommunication with main pump B, which withdraws equilibrating fluid fromreservoir A and delivers said fluid, under pressure, to column H. Aquick disconnect junction may optionally be included upstream of mainpump B in order to allow the end of tubing connecting reservoir A andmain pump B to be sealed to avoid or to minimize leakage and spillageshould either occur. Main pump B may be any suitable pump means,preferably embodied as a single-headed pump coupled to a pressure gaugeC, pressure sensor or transducer D, and pulse dampener E. Pressure atthe outlet of main pump B is monitored using pressure gauge C andpressure transducer D in combination with an internal pressure switchwhich shuts down the system if the pressure is above a predeterminedvalue. Main pump B should be capable of delivering the mobile phase ofthe chromatography system at a ripple-free, user-selected flow rate.When a single headed pump is used, a pulse dampener E is included tominimize ripples or pulsations in flow rate which may occur. Pulsedampener E preferably includes a pressurized tetrafluoroethylene (TFE)membrane which flexes during the delivery cycle of main pump B and whichis restored during the "refresh" cycle of pump operation. The pressureapplied to the membrane of pulse dampener E is dependent on the pressureat which main pump B is operated, being equal to about 80% of theoperating pressure of main pump B.

An inline filter F is placed downstream of pressure transducer D toremove particulate matter from the mobile phase. Preferably a filtercapable of removing particles greater than about 0.5 μm in diameter isemployed, though other filters may also be used in accordance with theinvention.

Downstream of inline filter F is an inlet valve X and an outlet valve Ywhich selectively establish a first flow path and a second flow path,outlet valve Y being in fluid communication with column H. In accordancewith the invention, the first flow path is in parallel to the secondflow path, and each flow path includes at least one injector valve.

Preferably, a plurality of manual injector valves is provided along thefirst flow path, as is exemplified in FIG. 9 by valves G₁, G₂, and G₃.The manual injector valves are preferably provided in series with eachother, wherein the outlet of each downstream valve is in fluidcommunication with the inlet of the adjacent upstream valve (e.g., theoutlet of valve G₁ is in fluid communication with the inlet of valve G₂and the outlet of valve G₂ is in fluid communication with the inlet ofvalve G₃). Sample or eluant may be added to the system through any ofmanual injector valves G₁, G₂, and G₃, in accordance with the presentembodiment. The manual injector valves of the first flow path may beused to determine the flow parameters of a specific chromatographicpurification empirically, prior to use of the automatic injection valvesK for the purification. The volumes accomodated by manual injectorvalves G₁, G₂, and G₃ may be the same or they may differ in accordancewith this embodiment. Each of manual injector valves G₁, G₂, and G₃ isfilled via a needle port in the valve handle assembly, which depictsseparate LOAD and INJECT positions. Fluid is injected into the manualinjector valve while the valve handle is in the LOAD position, using asyringe or other suitable cannula having a square end. The valve handleis rotated to the INJECT position to inject the fluid from the valveinto the flow path to column H.

Preferably, a plurality of automated injector valves K is provided inseries along the second flow path between X and Y, as is depicted inFIG. 9. Sample or eluant fluid is injected from reservoirs M into thesecond flow path using a pump L in fluid communication with eachautomated injector valve K, as exemplified in FIG. 9. Each pump L isunder the control of a suitable means, such as a computer program, whichdetermines the time of each injection and the flow rate of fluid pumped.A flowmeter may optionally be included between each pump L and therespective injector valve K to monitor fluid flow into each automatedinjector valve.

The column H employed in this embodiment is as described above inrelation to the column depicted in FIG. 3. A detector I is includeddownstream of column H to detect species that elute from column H. Aflowmeter J may optionally be included downstream of detector I, tomonitor flow of the mobile phase through the system. Similarly, valves N(which are preferably solenoid valves) are included downstream ofdetector I to divert the eluant stream to a collection vessel. A valve Pis also included downstream of detector I to divert the eluant flow intoa waste vessel.

While the apparatus of the present invention has been described in termsof a chromatographic column of packed particles, as described in theaforementioned U.S. patent application Ser. No. 08/552193, the columnsuseful in the present invention can also be in the alternative form of acapillary tube defining a hollow, elongated channel of substantiallyuniform internal diameter, the channel being provided with achromatographically active interior surface. The tube is formed suchthat turbulent flow will be induced in fluid pumped through the interiorat a velocity sufficient to create centrifugal forces in the fluid.

It has been found with apparatus of the present invention that acomplete cycle involving 100 mL sample plugs, using two different 5 mLeluant can be run within about 15 seconds, providing an extremely highthroughput. With the apparatus and method of the present invention, theamount of eluting solvent required for a given volume of sample, isgenerally considerably less. For example, for a 1×10 cm column with anominal volume of 8 mL, the amount of eluant used in the prior art istypically about 40 mL. For the present invention using a like column,this eluant volume is reduced to not more than about 8 mL, a substantialreduction by a factor of about 5 of often expensive eluant.

FIG. 13 demonstrates a preparative separation of α-chymotrypsinogen Afrom lysozyme, using the apparatus of the invention. For thisseparation, a cation exchange column equilibrated with phosphate buffer(pH 8) was employed. The proteins were sequentially eluted using varyingamounts of NaCl dissolved in phosphate buffer (pH 8), α-chymotrypsinogeneluting with 200 mM NaCl and lysozyme eluting with 2M NaCl. Afterseparation of the two proteins, a cleaning step was performed usingNaOH, allowing cyclic repetitions of the separation to be performed inaccordance with the present invention.

Since certain changes may be made in the above apparatus and processwithout departing from the scope of the invention herein involved, it isintended that all matter contained in the above description or shown inthe accompanying drawing shall be interpreted in an illustrative and notin a limiting sense.

What is claimed is:
 1. In chromatography apparatus including asubstantially uniform, elongated chromatography column containingchromatographically reactive surfaces, means for injecting into saidcolumn a discrete volume of liquid mixture containing at least onesolute that is reactive with said surfaces so as to load said column,and means for flowing eluant fluid through the loaded column, theimprovement wherein:said means for flowing said eluant fluid comprisesmeans for injecting at least one discrete plug of said eluant fluid intosaid column adjacent the input of said column so as to maintainminimized spatial step separation between said plug and said discretevolume of liquid mixture as said plug and volume traverse said columnwherein said column and said means for flowing are configured such thatthe flow of said volume of eluent traverses said column at a reducedvelocity greater than
 5000. 2. Chromatography apparatus as defined inclaim 1 wherein said means for injecting provides said plug traversingsaid column with a substantially plane front extending substantiallyperpendicularly to the axis of elongation of said column. 3.Chromatography apparatus as defined in claim 1 wherein said means forinjecting provides said plug traversing said column with a substantiallyplane end extending substantially perpendicularly to the axis ofelongation of said column.
 4. Chromatography apparatus as defined inclaim 1 wherein said means for injecting comprises a plurality ofindividual injectors for injecting respective plugs of said eluant fluidand said liquid mixture.
 5. Chromatography apparatus as defined in claim4 wherein said injectors are positioned for injecting a plurality ofdifferent plugs at respective spatially separated positions along anextension of the axis of elongation of said column so that said plugstraverse said column as closely bunched fluid plugs.
 6. Chromatographyapparatus as defined in claim 5 including means for controlling saidinjectors so that the latter operate for injecting said respective plugssubstantially simultaneously.
 7. Chromatography apparatus as defined inclaim 4 including means for controlling said injectors so that thelatter operate for injecting said respective plugs sequentially. 8.Chromatography apparatus as defined in claim 7 wherein said injectorsare positioned for injecting plurality of different plugs as sequentialinjections effected at one position located adjacent the input of saidcolumn.
 9. Chromatography apparatus as defined in claim 7 wherein saidinjectors are positioned for injecting a plurality of different plugs atrespective spatially separated positions along an extension of the axisof elongation of said column so that said plugs traverse said column asclosely bunched fluid plugs.
 10. Chromatography apparatus as defined inclaim 4 including means for cyclically repeating the injections of saidplugs of eluant fluid and mixture.
 11. Chromatography apparatus asdefined in claim 10 including means for effecting a flow of apredetermined volume of equilibrating fluid through said column betweencyclic repetitions of said injections.
 12. Chromatography apparatus asdefined in claim 10 wherein said means for injecting comprises at leastfirst and second injectors connected for injecting respective said plugsat respective first and second spatially separated positions along anextension of the axis of elongation of said column, and at least thirdand fourth injectors connected for injecting respective said plugs atthird and fourth spatially separated positions, said apparatus includingmeans for controlling the injections by said injectors for cyclicallyalternating injections of said respective plugs by said first and secondinjectors at said first and second positions, with injections of saidrespective plugs by said third and fourth injectors at said third andfourth positions.
 13. Chromatography apparatus as defined in claim 12wherein said third and fourth positions are the same as said first andsecond positions.